Voltage-gated sodium channels are transmembrane proteins which cause sodium permeability to increase. Depolarization of the plasma membrane causes sodium channels to open allowing sodium ions to enter along the electrochemical gradient creating an action potential.
Voltage-gated sodium channels are expressed by all electrically excitable cells, where they play an essential role in action potential propagation. They comprise a major subunit of about 2000 amino acids which is divided into four domains (D1-D4), each of which contains 6 membrane-spanning regions (S1-S6). The alpha-subunit is usually associated with 2 smaller subunits (beta-1 and beta-2) that influence the gating kinetics of the channel. These channels show remarkable ion selectivity, with little permeability to other monovalent or divalent cations. Patch-clamp studies have shown that depolarisation leads to activation with a typical conductance of about 20 pS, reflecting ion movement at the rate of 107 ions/second/channel. The channel inactivates within milliseconds (Caterall, W. A., Physiol. Rev. 72, S4-S47 (1992); Omri et al, J. Membrane Biol 115, 13-29; Hille, B, Ionic Channels in Excitable Membranes, Sinauer, Sunderland, Mass. (1991)).
Sodium channels have been pharmacologically characterised using toxins which bind to distinct sites on sodium channels. The heterocyclic guanidine-based channel blockers tetrodotoxin (TTX) and saxitoxin (STX) bind to a site in the S5-S6 loop, whilst xcexc-conotoxin binds to an adjacent overlapping region. A number of toxins from sea anemones or scorpions binding at other sites alter the voltage-dependence of activation or inactivation.
Voltage-gated sodium channels that are blocked by nanomolar concentrations of tetrodotoxin are known as tetrodotoxin sensitive sodium channels (Hille (1991) xe2x80x9cIonic Channels in Excitable Membranesxe2x80x9d, Sinauer Sunderland, Mass. (1991)) whilst sodium channels that are blocked by concentrations greater than 1 micromolar are known as tetrodotoxin-insensitive (TTXi) sodium channels (Pearce and Duchen Neuroscience 63, 1041-1056 (1994)).
Dorsal root ganglion (DRG) neurons express at least three types of sodium channels which differ in kinetics and sensitivity to TTX. Neurons with small-diameter cell bodies and unmyelinated axons (C-fibers) include most of the nociceptor (damage-sensing) population and express a fast TTX-sensitive current and a slower TTX-insensitive current. Of the five cloned sodium channel xcex1-subunit transcripts known to be present in dorsal root ganglia, none exhibits the properties of the TTX-insensitive channel.
Sodium channel blockers are used clinically to provide pain relief. Three classes of sodium channel blockers in common clinical use are: local anesthetics such as lidocaine, some anticonvulsants such as phenytoin and carbamazepine, and some antiarrhythmics such as mexiletine. Each of these is known to suppress ectopic peripheral nervous system discharge in experimental preparations and to provide relief in a broad range of clinical neuropathic conditions.
Applicants have now found a novel voltage-gated sodium channel (hereinafter referred to as a sodium channel specifically located in sensory neurons or also referred to as SNS sodium channel) that is present in sensory neurons (or neurones) but not present in glia, muscle, or the neurons of the sympathetic, parasympathetic, enteric or central nervous systems. Preferably the sodium channel of the invention is found in the neurons of the dorsal root ganglia (DRG) or cranial ganglia. More preferably the sodium channel of the invention is found in the neurons of the dorsal root ganglia. Preferably the sodium channel is specifically located in rat sensory neurons or human sensory neurons.
The sodium channel of the present invention is believed to play a role in nociceptive transmission because some noxious input to the central nervous system is known to be insensitive to TTX. Persistent activation of peripheral nociceptors has been found to result in changes in excitability in the dorsal horn associated with the establishment of chronic pain. Increased sodium channel activity has also been shown to underlie neuroma-induced spontaneous action potential generation. Conversely, chronic pain may be successfully treated by surgical or pharmacological procedures which block peripheral nerve activation. Blockage of nociceptor input may therefore produce useful therapeutic effects, even though central nervous system plasticity plays a pivotal role in the establishment of chronic pain. Sensory neuron-specific voltage-gated sodium channels, particularly sub-types associated with a nociceptive modality such as the sodium channel of the invention, thus provide targets for therapeutic intervention in a range of pain states. The electrophysiological and pharmacological properties of the expressed SNS sodium channel are similar to those described for the small diameter sensory neuron tetrodotoxin-resistant sodium channels. As some noxious input into the spinal cord is resistant to tetrodotoxin, block of expression or function of such a C-fiber-restricted sodium channel may have a selective analgesic effect.
In another aspect the present invention provides an isolated protein comprising a sodium channel specifically located in rat sensory neurons as encoded by the insert deposited in NCIMB deposit number 40744, which was deposited at The National Collections of Industrial and Marine Bacteria, 23 St Machar Drive, Aberdeen AB2 1RY, Scotland, United Kingdom on Jun. 27, 1995 in accordance with the Budapest Treaty.
The invention also provides nucleotide sequences coding for the SNS sodium channel. In a preferred embodiment, the nucleotide sequence encodes a sodium channel specifically located in rat sensory neurons which is as set out in FIG. 1a or a complementary strand thereof.
The approximately 6.5 kilobase (kb) transcript expressed selectively in rat dorsal root ganglia that codes for the novel sodium channel of the invention shows sequence similarities with known voltage-gated sodium channels. The cDNA codes for a 1,957 amino acid protein. In particular, the novel sodium channel of the invention shows 65% identity at the amino acid level with the rat cardiac tetrodotoxin-insensitive (TTXi) sodium channel. The aromatic residue that is involved in high-affinity binding of TTX to the channel atrium of TTX-sensitive sodium channels is altered to a hydrophilic serine in the predicted protein of the SNS sodium channel, whereas the residues implicated in sodium-selective permeability are conserved. The novel sodium channel specifically located in sensory neurons shows relative insensitivity to TTX (IC50 greater than 1 micromolar) and thus exhibits properties different from other cloned sodium channel transcripts known to be present in dorsal root ganglia.
The invention also provides expression and cloning vectors comprising a nucleotide sequence as hereinabove defined. In order to effect transformation, DNA sequences containing the desired coding sequence and control sequences in operable linkage (so that hosts transformed with these sequences are capable of producing the encoded proteins) may be included in a vector, however, the relevant DNA may then also be integrated into the host chromosome.
The invention also provides a screening assay for modulators of the sodium channel which is specifically located in sensory neurons wherein the assay comprises adding a potential modulator to a cell expressing the SNS sodium channel and detecting any change in activity of the sodium channel.
The present invention also provides a modulator which has activity in the screening assay hereinabove defined. Modulators of the sodium channel as hereinabove defined are useful in modulating the sensation of pain. Blockers of the sodium channel will block or prevent the trasmission of impulses along sensory neurons and thereby be useful in the treatment of acute, chronic or neuropathic pain.
The present invention thus relates to novel voltage-gated sodium channel proteins specific to sensory neurons, to nucleotide sequences capable of encoding these sodium channel proteins, to vectors comprising a nucleotide sequence coding for a sodium channel of the invention, to host cells containing these vectors, to cells transformed with a nucleic acid sequence coding for the sodium channel, to screening assays using the sodium channel proteins and/or host cells, to complementary stands of the DNA sequence which is capable of encoding the sodium channel proteins and to antibodies specific for the sodium channel proteins. These and other aspects of the present invention are set forth in the following detailed description.